1. Field of the Invention
This invention relates generally to the field of measuring gene expression. Specifically, the present invention relates to a novel method for directly quantifying gene expression by measuring RNA using capillary electrophoresis with laser-induced fluorescence.
2. Background Art
The analysis of gene expression is a common experiment conducted routinely in many laboratories. Profiling gene expression provides valuable insight into genes involved in normal cell/tissue homeostasis, organism development and information about genes, that when their expression is up or down-regulated, lead to disease. A number of methods are currently employed to examine gene expression with the most common being Northern blotting, RNase Protection Assay (RPA), and Real-time Polymerase Chain Reaction (RT-PCR). Each of these methods has advantages and disadvantages mainly relating to sample size, cost, time involved, use of radioactivity, and transcript information and sensitivity which is estimated to vary from 10,000 copies of mRNA (Northern blotting) to theoretically one copy (RT-PCR). However, all of these methods suffer from one common drawback. None of them provides a direct concentration or copy number of the mRNA. Instead, these methods provide only a relative measure of change. In most cases, information regarding changes in gene expression obtained by these methods involves relative changes in the intensity (either from densitometric analysis or fluorescence) which is normalized to an internal control gene with the data being presented as percent or fold increase/decrease compared to control. If a quantitative answer is desired, a standard curve must be generated using known concentrations of RNA, which may be difficult if the RNA is in short supply and time consuming. Several studies have used CE-LIF to examine the expression of a number of genes including leptin (Richards et al., 1999), cytokeratin 20 (van Eekelen, 2000) and glyceraldehyde-3-phosphate dehydrogenase (Fasco et al., 1994); however, in each of these cases the RNA was first subjected to reverse transcription followed by PCR, and the PCR product was then analyzed by CE-LIF.
Northern blotting is currently the only method which will provide information regarding transcript size. Both Northern blotting and RPAs can distinguish multiple transcripts; however, in the case of RPAs, this requires careful probe design. Both methods typically involve the use of radioactivity for maximal sensitivity of target RNA and typically involve 3-5 days before the result is obtained. RT-PCR is the fastest method currently available to examine gene expression, but it provides no information regarding transcript size, and examination of multiple transcripts cannot be obtained from a single RT-PCR reaction. In addition, in RT-PCR the target RNA is first converted to complementary DNA (cDNA), and then the amplification of the DNA is what is measured. While Northern blotting and RPA both directly detect the target RNA, both of these methods require the use of an internal standard, and neither provides a direct quantitative measure of the target RNA. RT-PCR is an indirect measure which is subject to problems of non-specific amplification or genomic DNA contamination. This method also requires the use of a standard for normalization, and the user must keep in mind that not all RNAs/DNAs are amplified at the same rate.
What is needed is a method of quantifying gene expression that is fast, sensitive, non-radioactive, and direct. The novel method disclosed herein combines many of the desired features of the above mentioned techniques, such as information regarding size of target and number of transcripts, and eliminates the need for radioactivity. Moreover, the described method provides a direct, quantitative measure of a target RNA.